Stem cell therapy is emerging as a beneficial strategy to treat patients with advanced heart disease. However, limited homing and survival of transplanted stem cells in the myocardium represents a barrier to therapeutic efficacy. During the initial term of funding, we focused on hypoxia preconditioning (HP) as a means to enhance homing and survival of cardiac stem cells by activating CXC chemokine receptor-4 (CXCR4) in HIF- 1?-dependent manner, which led to improved outcomes following cell transplantation in young mice. Unfortunately, aged C-MSC respond poorly to HP, suggesting that research into the molecular mechanisms that regulate HP in aged cells is needed to optimize this novel therapeutic strategy. Age-related impairments in tissue repair are associated with decreased neovascularization, a process that is regulated by HIF-1?- dependent signaling. Our preliminary data suggest that chromatin modifications in aged sca-1+ cardiac mesenchymal stem cells (C-MSC) may underlie impaired HIF-1? responses and compromise their function. Specifically, aged cells exhibit significantly greater histone 3 lysine 27 trimethylation (H3K27me3, a repressive chromatin modification) at the promoters of the HIF-1? target genes CXCR4 and IL-10, which are crucial regulators of stem/progenitor cell homing and survival. Moreover, aged cells exhibit increased expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb Repressor Complex 2 (PRC2). EZH2 is the only known enzyme capable of inducing H3K27me3 in mammalian cells. EZH2 may also cooperate with histone deacetylases (HDAC), which repress transcription by removing specific acetyl groups from histones. We hypothesize that in aged C-MSC, aberrant expression of EZH2 leads to repression of key HIF-1?-dependent genes through H3k27me3-induced gene silencing and by interacting with HDAC11, thus impairing HP responses and compromising therapeutic efficacy. We propose three aims to test our hypothesis. In Aim 1, we will determine the mechanisms whereby EZH2 is regulated by the transcription factor E2F1 in C-MSC, and we will target E2F1 in aged C-MSC to determine whether it is mechanistically linked to changes in EZH2 expression and function. In Aim 2, we will carry out genetic and biochemical experiments to determine whether HDAC11 functions as a part of the multi-protein EZH2-PRC2 complex that associates with the IL-10 promoter and represses HIF-1?-induced IL-10 gene expression in aged C-MSC. In Aim 3, we will ablate EZH2 in aged C-MSC to investigate functional responses to cell transplantation (i.e., cell homing, angiogenesis, cardiac repair). This will be the first i-depth study to investigate the role of epigenetic remodeling in regulating the function of aged stem cells. Our findings may lead to enhanced efficacy of cardiac stem cell therapy and identify new molecular targets that could improve cardiac repair in the elderly.